Biomarkers of Polycyclic Aromatic Hydrocarbon-DNA Damage And

Vol. 10, 581–588, June 2001 Cancer Epidemiology, Biomarkers & Prevention 581

Biomarkers of Polycyclic Aromatic Hydrocarbon-DNA Damage and Cigarette Smoke Exposures in Paired Maternal and Newborn Blood Samples as a Measure of Differential Susceptibility1

؎ Robin M. Whyatt,2 Wieslaw Jedrychowski, aromatic-DNA adduct levels (16.6 ؎ 12.5 versus 14.21 and plasma cotinine (0.002 ؍ Kari Hemminki, Regina M. Santella, Wei-Yann Tsai, 15.4/108 nucleotides; P Ke Yang, and Frederica P. Perera (14.2 ؎ 35.5 versus 8.3 ؎ 24.5 ng/ml; P < 0.001) but not ؎ ؎ Divisions of Environmental Health Sciences [R. M. W., R. M. S., F. P. P.] and for PAH-DNA adduct levels (7.9 9.9 versus 5.9 8.2/ When analyses were restricted .(0.13 ؍ Biostatistics [W-Y. T.], Mailman School of Public Health, Columbia 108 nucleotides; P University, New York, New York 10032; College of Medicine, Jagiellonian to the 80 mother/newborn pairs from whom the blood University, Krakow, Poland 31014 [W. J.]; and Department of Biosciences at sample was drawn concurrently (within1hofeach Novum, Karolinska Institute, Huddinge, Sweden S14157 [K. H., K. Y.] other), levels of all of the three biomarkers were significantly higher in the newborn compared with paired Abstract maternal blood samples (P < 0.05). Results suggest reduced detoxification capabilities and increased Human and experimental evidence indicates that the susceptibility of the fetus to DNA damage, especially in developing fetus may be more susceptible than the adult light of experimental evidence that transplacental to the effects of certain carcinogens, including some exposures to PAHs are 10-fold lower than paired polycyclic aromatic hydrocarbons (PAHs). Factors that maternal exposures. The results have implications for can modulate susceptibility include proliferation rates, risk assessment, which currently does not adequately detoxification capabilities, and DNA repair capacity. account for sensitive subsets of the population. Biomarkers can facilitate quantification of age-related susceptibility among human populations. In this study, we report on three biomarkers measured in paired blood Introduction samples collected at birth from 160 Polish mothers and Experimental and human evidence indicates that the developing newborns: 70 pairs from Krakow (a city with high air fetus and neonate can differ in susceptibility to chemical car- pollution including PAHs) and 90 pairs from Limanowa cinogenesis compared with the adult (reviewed in Refs. 1, 2). (an area with lower ambient pollution but greater indoor Age-related factors that may modulate susceptibility include coal use). Biomarkers were: WBC aromatic-DNA adducts differing rates of cell proliferation, abilities to activate and by 32P-postlabeling and PAH-DNA adducts by ELISA (as detoxify carcinogens, DNA repair capacity, and the number of indicators of DNA damage from PAHs and other target cells at risk (1, 2). Bioassays have shown the young to be aromatics) and plasma cotinine (as an internal dosimeter both more susceptible and more resistant, depending on the of cigarette smoke). Correlations were assessed by carcinogen tested, the species evaluated, the age at first expo- Spearman’s rank test, and differences in biomarker levels sure, and the target organ (1–5). A number of the PAHs3 are were assessed by the Wilcoxon signed-ranks test. A transplacental carcinogens in experimental bioassays, produc- significant correlation between paired newborn/maternal ing tumors in the liver, lung, lymphatic tissues, and nervous system of the offspring (4–6). Cancer incidence at these sites ؍ samples was seen for aromatic-DNA adduct levels (r P < 0.001) has been shown to be greater if the PAH exposure begins ;0.8 ؍ P < 0.001) and plasma cotinine (r ;0.3 prenatally or early postnatally compared with later in life, at .(0.13 ؍ P ;0.14 ؍ but not PAH-DNA adduct levels (r Among the total cohort, levels of the three biomarkers comparable doses (4, 7–10). However, human data on age- were higher in newborn samples compared with paired related susceptibility to PAH tumorigenesis are not available maternal samples. The difference was significant for (1). Biomarkers can facilitate evaluation of factors modulating susceptibility to carcinogens among human populations (11– 13). As an indication of DNA damage, carcinogen-DNA adducts represent a critical step on the carcinogenic pathway and Received 10/27/00; revised 3/8/01; accepted 3/14/01. are an informative biomarker of age-related susceptibility (11). The costs of publication of this article were defrayed in part by the payment of Therefore, we have compared levels of two biomarkers of DNA page charges. This article must therefore be hereby marked advertisement in damage from PAHs and other aromatics in paired maternal and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 32 1 Supported by Grant P50 ES09600, Grant RO1 ES08977, and Grant RO1 newborn blood samples: WBC aromatic-DNA adducts by P- ES06722 from the National Institute of Environmental Health Sciences; Grant postlabeling and PAH-DNA adducts by ELISA. Plasma cotin- R827027 from the United States Environmental Protection Agency; Grant DE- ine levels were also compared as an internal dosimeter of FG02-93ER61719 from the Department of Energy; and grants from the Gladys cigarette smoke exposure. and Roland Harriman Foundation, the March of Dimes Birth Defects Foundation, the W. Alton Jones Foundation, and the New York Community Trust. 2 To whom requests for reprints should be addressed, at Division of Environ- mental Health Sciences, Mailman School of Public Health, Columbia University, 60 Haven Avenue, B-1, New York, New York 10032. Phone: (212) 304-7273; 3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; NNK, Fax: (212) 544-1943; E-mail: [email protected]. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The biological basis for measuring DNA adducts derives Materials and Methods from extensive experimental data supporting their role in the Field studies were conducted in Poland during January–March, initiation and possibly in the progression of cancer (14). An 1992 under the direction of Dr. Wieslaw Jedrychowski (Jagiel- association between PAH-DNA adduct levels and cancer risk lonian University, Krakow, Poland) in accordance with current has been seen previously (14–17) in both epidemiological and guidelines for human subjects. Enrollment was restricted to experimental research. PAHs such as benzo(a)pyrene readily women who had resided in Krakow or Limanowa for at least 1 cross the placenta. Experimental studies (18–20) using radio- year and was limited to vaginal deliveries. In all of the cases, labeled PAHs indicate that the dose to the fetus is generally an samples of umbilical cord blood (20–60 ml) were collected at order of magnitude or more lower than the dose to paired delivery, and a maternal blood sample was collected within an maternal tissues. Adducts formed between DNA and various average of 8.7 Ϯ 11.6 h after delivery (range, 5 h before PAHs have been measured in multiple fetal tissues in experi- delivery to 66 h after delivery). For a subset of 80 of these mental rodent bioassays (21–23). Fetal levels are generally mother/newborn pairs, the maternal blood sample was drawn lower than maternal levels but higher than expected given the within1hofdelivery (either before or after delivery). Samples maternal/fetal dose differential. Levels of genotoxic damage were processed as described previously (44). (micronuclei formation and DNA single strand breaks) after A detailed, validated questionnaire administered to the transplacental PAH exposure have been shown experimentally mother within 2 days postpartum included information on to be higher in fetal than paired maternal tissues (23–25). One smoking (active and passive), residential and employment his- prior molecular epidemiological study has measured PAH- tories, use of coal stoves for residential heating, and other DNA adducts in maternal and cord blood samples (26). When environmental exposures as described previously (44). measured by ELISA with fluorescence detection, PAH-DNA Aromatic-DNA Adducts. The 32P-postlabeling TLC assay adducts were detected in 18 of 27 and 13 of 21 of the maternal was carried out at the Karolinska Institute as described previ- and newborn WBCs, respectively. For mothers and newborns ously (50, 51). In brief, DNA was extracted from the crude with detectable adducts, mean levels were similar (26). nuclei using organic solvents after degrading RNAs and pro- ␮ Cotinine is the major proximal metabolite of nicotine but teins; 5 g of DNA was digested by micrococcal nuclease and Ј has a longer half-life in sera (15–40 h versus 1–3 h for nico- spleen phosphodiesterase to 3 nucleotides. Adducts were then tine), and serum concentrations of cotinine are 10-fold higher enriched by nuclease P1 treatment. A postlabeling reaction was than they are for nicotine (27–31). It is less biologically active carried out and applied on a TLC plate for adduct separation in than nicotine (30, 31) but has been widely used as a marker of three dimensions. After autoradiography, the adduct spots were exposure to tobacco smoke (29, 32, 33), which contains a excised from the successfully developed TLC plate for counting of radioactivity as uniform areas covering most of the radioac- myriad of carcinogens, including the nicotine-derived carcino- tive fractions of the plate (51). Two to five assays were carried gens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) out for each sample, and the mean of all of the assay results and NЈ-nitrosonornicotine (34, 35), as well as carcinogenic were calculated. The detection limit of the assay was 0.07 PAHs (27). Nicotine readily crosses the placenta, and both adducts/108 nucleotides. The assay detects multiple PAHs and nicotine and cotinine have been measured in fetal tissues (30, other aromatic compounds bound to DNA (52). Aromatic-DNA 36–38). Studies (39–43) comparing paired maternal/fetal co- adduct levels were determined for 122 mother/newborn pairs. tinine levels are few and have yielded conflicting results. Both higher and lower concentrations in maternal samples compared PAH-DNA Adducts. PAH-DNA adducts were measured by a competitive ELISA with fluorescence end-point detection es- with fetal samples have been reported. sentially as described previously (53). The detection limit of the This study extends prior evaluations in the current cohort assay is two adducts/108 nucleotides. Samples were assayed in of 70 mother/newborn pairs from Krakow, Poland (an industrial triplicate at 50 ␮g of DNA/well. The median values were used city with elevated ambient air pollution including PAHs from to determine the percentage of inhibition. When sufficient DNA coal-burning for industry and residential heating) and 90 pairs was available (63% of samples), the assay was repeated. Lab- from Limanowa (a small town located 70 km southeast of oratory personnel were blinded to subject status. The antiserum Krakow with lower ambient pollution levels but 2-fold more was elicited against benzo(a)pyrene-diol-epoxide-DNA but frequent use of coal-stoves for indoor home heating (44). We recognizes other structurally related PAH diol-epoxide-DNA have reported previously (45, 46) that a significant association adducts, including those formed by benz[a]anthracene and Ͻ ␮ was seen between ambient particulate levels of 10 m (PM10) chrysene (54). Thus, positive reaction with the antiserum may and PAH-DNA but not aromatic-DNA adduct levels in both indicate the presence of multiple PAH-DNA adducts in the maternal and newborn WBCs. Maternal active and passive sample. Values are expressed as the amount of BPDE-DNA cigarette smoking status was significantly associated with ma- that would cause a similar inhibition in the assay. PAH-DNA ternal PAH-DNA but not aromatic-DNA adduct levels (45, 46). adduct levels were determined for 112 mother/newborn pairs. However, adduct levels in maternal WBCs did not differ sig- Plasma Cotinine. The method involved liquid/liquid extrac- nificantly between ex-smokers and nonsmokers (45, 46). Nor tion of plasma, followed by gas chromatographic separation was any association seen between maternal smoking status and using a 30-m 0.25 ␮ DB WAX megabore column and a nitro- either measure of DNA adducts in newborn WBCs, possibly gen phosphorus detector operated in the nitrogen mode as because the activity of placental enzymes that are induced by described previously (44). An internal standard, N-methyl co- maternal smoking reduced the biologically effective dose to the tinine, was added to the plasma before extraction. Five-point fetus (47, 48). Newborn PAH-DNA adduct levels were signif- standard curves were generated for each analytical run, and icantly inversely associated with birth weight, length, and head low- and high-quality control samples were processed each day. circumference (49). Newborn plasma cotinine was inversely The method requires cold trapping injection followed by tem- associated with birth weight and length (49). The present report perature programming to achieve optimal separation resulting extends the research to compare levels of three biomarkers in in an analysis time of 18 min/sample. To facilitate productivity, mother/newborn pairs. an autosampler and online automatic data reduction were used

Table 1 Number of mother/newborn pairs,a maternal age,b and smoking smoking status, newborn adduct levels again exceeded maternal statusa of mothers levels among both nonsmokers and ex-smokers, but the difference was significant only among the nonsmokers (P ϭ 0.005) Mother/newborn pairs 160 and was not significant among the ex-smokers (P ϭ 0.2). By Mother’s age (yr) 26.3 Ϯ 4.8 Current smokers 16 (10%) contrast, aromatic-DNA adduct levels among newborns of cur- Ex-smokers 38 (24%) rent smokers were similar and not significantly different from Nonsmokers 106 (66%) paired maternal levels (P ϭ 0.79; Table 2). a When analyses were restricted to mother/newborn pairs Number of subjects (%). ϭ b Mean Ϯ SD. from whom blood was drawn concurrently (n 61), newborn aromatic-DNA adduct levels consistently exceeded maternal adduct levels, but the difference was significant only when analyses involved all of the mother/newborn pairs with con- so that samples could be processed during the evening or current blood sampling (P ϭ 0.04) and was not significant overnight, as needed. Cotinine levels were determined on 158 when analyses were stratified by smoking status (P Ն 0.1). 8 mother/newborn pairs, including 80 pairs from whom blood Table 3 compares PAH-DNA adduct levels (/10 nucleo- samples were drawn within1hofeach other (concurrent tides) in mother/newborn pairs. When analyses involved all of samples). the mother/newborn pairs (n ϭ 112), PAH-DNA adduct levels Statistical Analyses. Means and SDs for biomarker levels in were somewhat higher in newborn compared with paired ma- paired maternal and newborn samples are presented. Statistical ternal WBCs among the total cohort and among newborns of nonsmokers and ex-smokers (Table 3). The difference was of analyses were undertaken using nonparametric statistics be- ϭ cause of the distributional properties of the biomarkers and the borderline significance among the nonsmokers (P 0.05; large number of samples that were below the limit of detection. Table 3). Among the 10 current smokers, maternal adduct Correlations between biomarkers in paired maternal and new- levels exceeded the newborn levels in 7 of the 10 pairs, although they were less than the newborn levels in 1 of the 10 born samples were assessed by Spearman’s rank. The differ- ϭ ences in biomarker levels in mother/newborn pairs were as- pairs (P 0.05; Table 3). When analyses were restricted to the mother/newborn pairs from whom the blood samples were sessed by the Wilcoxon signed-ranks test. Analyses were first ϭ undertaken involving all of the mother/newborn pairs (total drawn concurrently (n 60), PAH-DNA adducts were higher sample) and then were restricted to the 80 mother/newborn in the newborn compared with paired maternal WBCs in all of pairs from whom blood samples were drawn within1hofeach the groups except current smokers. The difference was significant for the total cohort (P ϭ 0.04) and among nonsmokers other (concurrent samples). Associations were considered sig- ϭ nificant at P Յ 0.05. (P 0.03). Table 4 compares plasma cotinine levels (ng/ml) in maternal and newborn blood samples for all of the mother/ Results newborn pairs and for the mother/newborn pairs from whom Data on demographic variables and smoking status are sum- blood was drawn concurrently. Newborn cotinine levels con- marized in Table 1. Fig. 1 and Fig. 2 show the correlation sistently exceeded maternal levels, and in most cases the dif- between biomarkers in paired maternal and newborn blood ference was highly significant. Specifically, as seen in Table 4, samples for all of the mother/newborn pairs. The correlation among the total cohort (n ϭ 158), newborn levels exceeded between WBC PAH-DNA adduct levels in paired maternal/ those in the paired maternal sample five times as frequently as newborn samples was not significant (Fig. 1A; r ϭ 0.14; P ϭ maternal levels exceeded the paired newborn levels (see ranks, 0.13). WBC aromatic-DNA adduct levels were moderately but Table 4; P Ͻ 0.001). Similarly, among the concurrent samples significantly correlated (Fig. 1B; r ϭ 0.3; P Ͻ 0.001). Plasma (n ϭ 80; Table 4), newborn cotinine levels again exceeded the cotinine levels were highly correlated (Fig. 2A; r ϭ 0.8; P Ͻ paired maternal levels five times as frequently as the maternal 0.001). When analyses were restricted to the mother/newborn levels exceeded the paired newborn levels, a difference that pairs from whom the blood samples were drawn concurrently, remained highly significant despite the reduced sample size the correlations between the biomarkers in paired samples (P Ͻ 0.001). remained essentially unchanged. Specifically, WBC PAH- After stratifying by smoking status, newborn cotinine lev- DNA adduct levels in paired samples were not correlated (r ϭ els exceeded paired maternal levels in all of the strata (non- 0.08; P ϭ 0.53; n ϭ 60). WBC aromatic-DNA adduct levels in smokers, ex-smokers, and current smokers). This difference paired samples were moderately correlated (r ϭ 0.3; P ϭ 0.01; was significant (P Յ 0.005) in all of the strata when analyses n ϭ 61). Plasma cotinine levels in paired samples were highly involved the total cohort (Table 4). When analyses were re- correlated (r ϭ 0.8; P Ͻ 0.001; n ϭ 80; data not shown). stricted to the concurrent samples (Table 4), newborn cotinine Tables 2–4 compare the biomarker levels in mother/ levels exceeded paired maternal levels in all of the strata newborn pairs and provide results of the Wilcoxon signed-ranks (nonsmokers, ex-smokers, and current smokers), and the mag- test. Analyses are provided both for the total cohort and for the nitude of the difference was similar to that seen in the total mother/newborn pairs from whom the blood samples were cohort. However, the difference was significant only among drawn concurrently. nonsmokers (P ϭ 0.008; Table 4) and was not significant As seen from Table 2, aromatic-DNA adduct levels (/108 among ex-smokers and current smokers, because of the reduced nucleotides) were higher in newborn than in paired maternal sample size (n ϭ 16 and n ϭ 4 pairs, respectively). WBCs. Specifically, when analyses involved all of the mother/ Table 5 shows the number of mother/newborn pairs with newborn pairs (n ϭ 122), newborn aromatic-DNA adduct lev- biomarker levels above and below the limit of detection. The els exceeded paired maternal levels approximately twice as ratio (mean Ϯ SD, range) of newborn/maternal biomarker lev- frequently because they were less than paired maternal levels els for the mother and newborn pairs in which the biomarker in (see ranks for mother/newborn pairs; Table 2), a difference that both the maternal and newborn blood samples was above the was statistically significant (P ϭ 0.002). After stratifying by limit of detection is also presented. As seen, the levels of

Fig. 1. Correlation between DNA adduct levels/108 nucleotides in paired maternal and newborn WBCs. A, correlation between maternal and newborn WBC PAH- DNA adducts; r ϭ 0.14; P ϭ 0.13. B, correlation between maternal and newborn WBC aromatic-DNA adduct levels; r ϭ 0.3; P Ͻ 0.001.

aromatic DNA-adducts were above the limit of detection in the limit of detection, there was a fairly equal distribution both maternal and newborn WBCs for 113 of 122 (93%) of the among the pairs in which the adduct levels in both the maternal mother/newborn pairs. Among these pairs, newborn aromatic- and newborn WBCs were below the limit of detection (n ϭ 24) DNA adduct levels averaged 4.1 Ϯ 12.5 (range, 0.04–105.3) compared with the pairs in which: (a) the adduct levels were times higher than paired maternal adduct levels. above the limit of detection in the newborn WBCs and below By contrast, the levels of PAH-DNA adducts were above the limit of detection in the maternal WBCs (n ϭ 20); and (b) the limit of detection in both the maternal and newborn WBCs the adducts were below the limit of detection in the newborn in only 44 of 112 (39%) of the mother/newborn pairs (Table 5). WBCs and were above the limit of detection in the maternal Among these mother/newborn pairs with adducts above the WBCs (n ϭ 24). limit of detection, newborn PAH-DNA adduct levels averaged Levels of plasma cotinine were above the limit of detec- 2.7 Ϯ 3.2 (range, 0.11–15.5) times higher than paired maternal tion in both the maternal and newborn blood samples in 58 of adduct levels. Among the 68 of 122 (61%) of the mother/ 158 (38%) of the mother/newborn pairs (Table 5). Among these newborn pairs in which the PAH-DNA adduct levels in either pairs, newborn plasma cotinine averaged 2.0 Ϯ 1.8 (range, or both of the maternal and/or the newborn WBCs were below 0.67–9.7) times higher than paired maternal cotinine levels. For

Fig. 2. Correlation between plasma cotinine levels (ng/ml) in paired maternal and newborn blood samples. A, correlation between plasma cotinine levels in mother/newborn pairs; r ϭ 0.8; P Ͻ 0.001.

Table 2 WBC aromatic-DNA adduct levels by 32P-postlabeling paired maternal and newborn blood samples

Ranks for mother/newborn pairsa Mean Ϯ SD/108 nucleotides Negative ranksb Positive ranksc Tiesd Pa Maternal Newborn No.e Sumf No.e Sumf No.e A. WBC aromatic-DNA adduct levels in all of the mother/newborn pairs (total sample) Total cohort (n ϭ 122) 14.2 Ϯ 15.4 16.6 Ϯ 12.5 41 2430 79 4830 2 P ϭ 0.002 Nonsmoker (n ϭ 79) 15.0 Ϯ 16.3 17.1 Ϯ 11.3 25 976 53 2104 1 P ϭ 0.005 Ex-smoker (n ϭ 32) 13.0 Ϯ 15.6 15.7 Ϯ 14.9 11 185 20 311 1 P ϭ 0.22 Current smoker (n ϭ 11) 12.0 Ϯ 4.2 14.7 Ϯ 13.7 5 30 6 36 0 P ϭ 0.79 B. WBC aromatic-DNA adduct levels in mother/newborn pairs with blood drawn concurrently (concurrent samples) Total cohort (n ϭ 61) 15.2 Ϯ 17.8 17.8 Ϯ 14.4 22 655 39 1237 0 P ϭ 0.04 Nonsmoker (n ϭ 46) 16.6 Ϯ 20.1 17.8 Ϯ 12.4 18 390 28 692 0 P ϭ 0.10 Ex-smoker (n ϭ 12) 10.6 Ϯ 6.9 16.5 Ϯ 20.4 4 29 8 49 0 P ϭ 0.43 Current smoker (n ϭ 3) 10.9 Ϯ 2.2 23.5 Ϯ 20.1 0 0 3 6 0 P ϭ 0.11 a Wilcoxon signed-ranks test. b Newborn adduct level Ͻ maternal adduct levels. c Newborn adduct levels Ͼ maternal adduct levels. d Newborn adduct levels ϭ maternal adduct levels. e Number of mother/newborn pairs. f Sum of ranks.

73 of 158 (46%) of the pairs, cotinine in both maternal and ing fetus to DNA damage from this class of carcinogens. newborn plasma was below the limit of detection. Among the Experimental evidence in rodents indicates that transplacen- 27 pairs in which cotinine in either the maternal or the newborn tal exposures to PAHs are generally an order of magnitude or plasma was below the limit of detection, there was a higher more lower than paired maternal exposures (18). In light of proportion of pairs in which the cotinine level was above the these experimental results, our findings suggest that the limit of detection in the newborn and below the limit of detec- amount of DNA damage/delivered dose of PAHs may be tion in the mother (22 of 27) than pairs in which the cotinine considerably higher in the fetus relative to his/her mother. was below the limit of detection in the newborn and was above This increased susceptibility could result from reduced de- the limit of detection in the mother (5 of 27). toxification capabilities or decreased DNA repair capacity during fetal development (reviewed in Refs. 1, 55). Discussion The ratio of DNA damage in newborn WBCs compared This study found that the levels of DNA damage from PAHs with maternal WBCs differed by maternal smoking status. and other aromatics were higher in newborn WBCs than in Specifically, among newborns of nonsmoking women, both paired maternal WBCs. The difference in mean adduct levels aromatic-DNA adducts and PAH-DNA adducts were higher in between newborn and maternal WBCs was small and not newborn WBCs compared with paired maternal WBCs, a dif- always statistically significant; nonetheless, the results point ference that was highly significant for aromatic-DNA adducts to the possibility of increased susceptibility of the develop- (P ϭ 0.005) and of borderline significance for PAH-DNA

Table 3 WBC PAH-DNA adduct levels by ELISA in paired maternal and newborn blood samples

Ranks for mother/newborn pairsa Mean Ϯ SD/108 nucleotides Negative ranksb Positive ranksc Tiesd Pa Maternal Newborn No.e Sumf No.e Sumf No.e A. WBC PAH-DNA adduct levels in all of the mother/newborn pairs (total sample) Total cohort (n ϭ 112) 5.9 Ϯ 8.2 7.9 Ϯ 9.9 43 1557 44 2271 25 P ϭ 0.13 Nonsmoker (n ϭ 70) 5.1 Ϯ 6.3 8.1 Ϯ 9.4 25 498 28 933 17 P ϭ 0.05 Ex-smoker (n ϭ 32) 5.4 Ϯ 6.7 8.4 Ϯ 1.3 11 130 15 221 6 P ϭ 0.25 Current smoker (n ϭ 10) 12.9 Ϯ 17.5 4.6 Ϯ 8.4 7 32 1 4 2 P ϭ 0.05 B. WBC PAH-DNA adduct levels in mother/newborn pairs with blood drawn concurrently (concurrent samples) Total cohort (n ϭ 60) 5.7 Ϯ 7.4 9.4 Ϯ 11.0 21 382 27 794 12 P ϭ 0.04 Nonsmoker (n ϭ 45) 4.9 Ϯ 6.8 9.1 Ϯ 10.2 15 191 21 475 9 P ϭ 0.03 Ex-smoker (n ϭ 12) 9.4 Ϯ 9.5 12.7 Ϯ 14.0 5 24 6 42 1 P ϭ 0.42 Current smoker (n ϭ 3) 3.7 Ϯ 4.6 1.0 Ϯ 0.011002P ϭ 0.32 a Wilcoxon signed-ranks test. b Newborn adduct level Ͻ maternal adduct levels. c Newborn adduct levels Ͼ maternal adduct levels. d Newborn adduct levels ϭ maternal adduct levels. e Number of mother/newborn pairs. f Sum of ranks.

Table 4 Plasma cotinine levels in paired maternal and newborn blood samples

Ranks for mother/newborn pairsa Mean Ϯ SD ng/ml Negative ranksb Positive ranksc Tiesd Pa Maternal Newborn No.e Sumf No.e Sumf No.e A. Plasma cotinine levels in all of the mother/newborn pairs (total sample) Total cohort (n ϭ 158) 8.3 Ϯ 24.5 14.2 Ϯ 35.5 13 344 64 2659 81 P Ͻ 0.001 Nonsmoker (n ϭ 104) 3.2 Ϯ 16.5 4.0 Ϯ 18.3 11 201 33 790 60 P ϭ 0.001 Ex-smoker (n ϭ 38) 11.4 Ϯ 28.2 17.7 Ϯ 38.3 2 21 16 150 20 P ϭ 0.005 Current smoker (n ϭ 16) 34.7 Ϯ 38.6 72.2 Ϯ 53.8 0 0 15 120 1 P ϭ 0.001 B. Plasma cotinine levels in mother/newborn pairs with blood drawn concurrently (concurrent samples) Total cohort (n ϭ 80) 7.2 Ϯ 25.9 7.7 Ϯ 27.3 8 159 33 703 39 P Ͻ 0.001 Nonsmoker (n ϭ 60) 3.3 Ϯ 19.4 3.4 Ϯ 18.9 7 104 23 362 30 P ϭ 0.008 Ex-smoker (n ϭ 16) 9.4 Ϯ 22.9 10.1 Ϯ 23.9 1 7 7 29 8 P ϭ 0.12 Current smoker (n ϭ 4) 57.0 Ϯ 62.8 63.7 Ϯ 71.4 0 0 3 6 1 P ϭ 0.11 a Wilcoxon signed-ranks test. b Newborn cotinine levels Ͻ maternal cotinine levels. c Newborn cotinine levels Ͼ maternal cotinine levels. d Newborn cotinine levels ϭ maternal cotinine levels. e Number of mother/newborn pairs. f Sum of ranks.

Table 5 Number of maternal/newborn pairs with biomarker levels above and below the limit of detection and ratio of newborn/maternal biomarker levels among pairs with detectable levels

Pairs with maternal and/or newborn levels below limit Pairs with maternal and newborn levels above of detection the limit of detection Both below Newborn above/ Newborn below/ Both above Ratio newborn/maternal detection maternal below maternal above detection levels: mean Ϯ SD (range) Aromatic-DNA adduct levels N ϭ 2 N ϭ 2 N ϭ 5 N ϭ 113 4.1 Ϯ 12.5 (0.04–105.3) PAH-DNA adduct levels N ϭ 24 N ϭ 20 N ϭ 24 N ϭ 44 2.7 Ϯ 3.2 (0.11–15.5) Plasma cotinine N ϭ 73 N ϭ 22 N ϭ 5 N ϭ 58 2.0 Ϯ 1.8 (0.67–9.7)

adducts (P ϭ 0.05). By contrast, among newborns of current have shown placental CYP1A1 to be highly induced by mater- smoking women, aromatic-DNA adducts were not significantly nal cigarette smoking. Prior evidence suggests that induction of different from paired maternal levels (P ϭ 0.8) and PAH-DNA CYP1A1 in placentas of smokers may reduce the biologically adducts were lower (P ϭ 0.05). It is possible that enzyme effective dose to the fetus (47, 48). As reported previously (45, activity of cytochrome P4501A1 (CYP1A1) in placental tissue 60), we also found that placental CYP1A1 was inversely, contributed to these differences. We and others (44, 56–59) although not significantly, correlated with PAH-DNA adduct

levels in WBCs and placental tissue from newborns in the The findings have implications for risk assessment, which cur- current cohort. rently does not adequately account for sensitive subsets of the This study also found that cotinine levels were consis- population. tently and, in most cases, significantly higher in newborn plasma compared with paired maternal plasma. Few prior studies (39–43) have compared cotinine levels in mother/newborn Acknowledgments pairs, and results have been conflicting, with both higher and We thank T. Randall, Dr. A. Rundle, Dr. T. L. Young, T. Cooper, Q. Wang, D. Allen, F. Crofts, and R. Lonow for their assistance. lower concentration in the neonate reported. It is likely that methodological differences in the timing of the maternal blood collection relative to newborn blood collection have contrib- References uted to these differences, because most prior studies have not 1. Anderson, L. M., Diwan, B. A., Fear, N. T., and Roman, E. Critical windows collected the maternal and newborn blood samples concur- of exposure for children’s health: cancer in human epidemiological studies and rently. Given the short half-life of cotinine in plasma, concur- neoplasms in experimental animal models. Environ. Health Perspect., 108: 573– 594, 2000. rent sampling is needed to differentiate the possibilities that 2. National Academy of Sciences. Pesticides in the Diets of Infants and Children. newborn/maternal cotinine ratios reflect a true biological dif- Carcinogenesis and Mutagenesis, pp. 70–76. Washington, DC: National Acad- ference in dose and/or metabolic processing of cotinine or a emy Press, 1993. difference in elapsed time between cigarette smoke exposure 3. Vesselinovitch, S. C., Kandala, D. R., and Mihailovich, N. Neoplastic response and newborn versus maternal blood collection. 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Robin M. Whyatt, Wieslaw Jedrychowski, Kari Hemminki, et al.

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